Bacillus anthracis, the causative agent of anthrax, has become a serious bioterrorism threat. Due to the highly virulent nature of this microorganism, it is crucial that antibiotic therapy begins prophylactically, therefore effective drugs need to be inexpensive and tolerated in a large population. Inhibitors of dihydrofolate reductase (DHFR) have been used for decades to treat bacterial infections, however, trimethoprim, a clinically used DHFR inhibitor, is ineffective against B. anthracis due to poor interactions with the Bacillus enzyme. However, alternative DHFR inhibitors that exhibit high potency against the Bacillus enzyme should be excellent therapeutics to treat anthrax infections. We have designed a number of new DHFR inhibitors and found that several inhibit DHFR from B. anthracis and promote B. anthracis cell death while maintaining low mammalian cell toxicity. Novel methods for the syntheses of these compounds have resulted in an inhibitor with a submicromolar IC50 value and a MIC99 value of 20 5g/mL with no mammalian cell toxicity. Recent efforts have delivered more potent compounds with MIC99 values as low as 3.5 5g/mL. The goal of the work in this application is to develop a lead and a backup compound that are potent and selective inhibitors of B. anthracis growth. To do this, we have four specific aims: one, synthesize a focused library of inhibitors intended to increase potency and determine resistance generation; two, build on the potent inhibitors to synthesize a library of selective inhibitors; three, determine the crystal structure of Bacillus anthracis DHFR bound to a lead compound in order to evaluate protein:ligand interactions; and four, develop and synthesize advanced leads based on results from protein binding and P450 enzyme activity assays as well as additional crystal structures of resistant proteins. PUBLIC HEALTH RELEVANCE: There is a critical need for new antibiotics effective against anthrax infections. Small molecule inhibitors of a key metabolic enzyme have been synthesized and proven to be potent without being toxic to mammalian cells. The goal of this proposal is to use the structure of the enzyme to further develop those compounds to effectively inhibit the growth of Bacillus anthracis Sterne at low concentrations.